PROJECT SUMMARY Significance. Acute lung injury (ALI) due to lung infection is a major cause of mortality and morbidity. The role of resident alveolar macrophages (AMs) in the pathology remains inadequately understood because of the lack of in situ studies. We will focus on sessile alveolar macrophages (SAMs), which we recently identified as alveolus-adherent, resident AMs. Since SAMs generate cytosolic Ca2+ (cCa2+) increases after LPS exposure, one major goal is to evaluate the significance of these cCa2+ responses as determinants of progressive mitochondrial dysfunction during development of ALI. Another major goal is to define how SAMs integrate with alveolar function as regards pathogen delivery and micromechanical interactions with the alveolar epithelium. New mechanistic understanding of ALI will be achieved. Specific Aims. The specific aims are first, to determine the role of mitochondrial Ca2+ buffering (MCB) in SAMs as a mechanism by which cCa2+ increases determine increases of mitochondrial Ca2+ (mCa2+) and mitochondrial H2O2 production. Second, to determine the role of alveolar wall liquid and alveolar surfactant as mechanisms of pathogen delivery to SAMs, and the extent of alveolar mechanical interactions with SAMs. Approach. We will achieve these aims by live confocal microscopy of isolated perfused mouse lungs in conjunction with flow cytometry of SAMs extracted from lung tissue and from bronchoalveolar lavage (BAL), histology and survival studies. Our determinations will include: (1) cCa2+ and mitochondrial Ca2+ in SAMs of intact alveoli; (2) assays of MCB and of mROS; (3) assaying role of the mitochondrial Ca2+ uniporter (MCU) in Ca2+ regulation in SAMs through use of specific mutants, siRNA and mice; (4) expression of light-activated probes to optogenetically induce cCa2+ increase and generate mitochondrial H2O2; (5) Expression of a tension sensor to detect SAM dimensional changes during lung stretch. Impact. This proposal will lead to the first systematic understanding of the role of mitochondria as determinants of macrophage function in situ. The extent to which loss of the macrophage MCU exacerbates ALI, and the extent to which MCU reinstatement in SAMs promote lung repair will be defined. Mechanisms underlying pathogen delivery to AMs will be understood for the first time. New understanding achieved may contribute to development of macrophage based cell therapy for ALI.